We propose to extend the classic Jost hypothesis of sexual differentiation by showing that female sexual differentiation is not simply a default pathway secondary to a lack of androgen but an active process dependent upon estrogen. Furthermore, male external genitalia (ExG) differentiation may not be solely a function of androgens but also influenced by estrogens. Thus, the overall hypothesis of this proposal is that normal male and female development of the external genitalia is dependent upon the correct balance of androgen and estrogen that signal through their respective receptors. Our recent studies on a variety of mutant mice (?ERKO, NERKI, ER? null, ?ERKO, and AROM+) demonstrate profound effects of these "estrogen mutants" on clitoral development, and subtle effects on development of the penis. Given the wealth of our Preliminary Data, we are in an excellent position (a) to establish an important role of estrogen in normal clitoral (a new concept) and possibly normal penile development, (b) to identify specific morphogenetic events in the developing external genitalia that are dependent upon estrogen (especially in the epithelium), and (c) to demonstrate that female sex differentiation of the external genitalia is in fact a unique active process requiring ER? signaling and not simply a default pathway associated with the absence of androgen. To validate the hypothesis that normal female and male development of the mouse external genitalia are dependent upon signaling through ER?, the following Specific Aims will be pursued: 1. Define ontogeny of sex differentiation of male and female ExG and hormonal parameters of sex differentiation of the ExG. 2. Identify morphological features within the male and female external genitalia that are directly or indirectly dependent upon estrogens through analysis of mutant mice. 3. Define the role of proliferation and apoptosis in development of sexual dimorphism. 4. High resolution localization of AR and ER? in the developing male and female external genitalia. 5. Define the role of epithelial ER? in the development of the external genitalia. This proposal will extend the classic Jost hypothesis adding estrogen along with androgen as a critical hormone for normal development. The concepts emerging from this investigation will also have translational relevance to a variety of disorders of sexual differentiation in humans including hypospadias. For example, abnormalities such as hypospadias might be explained by a disruption in the normal balance between androgens and estrogens. If this is the case then prevention strategies could be designed for at risk populations to prevent abnormal prenatal exposure to estrogenic compounds. PUBLIC HEALTH RELEVANCE: We propose to validate the paradigm that estrogen, in addition to the classic male hormone testosterone, is critical for normal feminine development of the clitoris and possibly masculine development of the penis by demonstrating actual morphogenetic/cell differentiation events attributable to either estrogen or androgen action. Understanding this new role for estrogen in sexual differentiation is germane to understanding the etiology of hypospadias and other disorders of sex development.